1. Field of the Invention
This invention relates to interactions between biological molecules, particularly proteins, and methods for detecting and quantifying said interactions. The invention is particularly related to detection of protein—protein interactions that occur in the cellular cytoplasm. The invention specifically provides methods and reagents for detecting said cytoplasmic protein—protein interactions using a GAL regulatory factor in yeast, Gal80p, and the yeast galactose regulon. The invention further provides methods for the detection of nuclear export sequences and nuclear localization sequences. This invention also relates to the inducible production of proteins from GAL gene promoters without requiring galactose as the inducing molecule. The invention specifically provides methods and reagents for inducing Gal4p-mediated GAL gene promoter expression by small molecules other than galactose.
2. Background of the Related Art
The Human Genome Project has recently revealed the complete human genetic sequence. From studies on only a very small fraction of the genome (likely less than 0.1%), it appears that protein—protein interactions most often comprise key mechanistic features of biological processes. Protein—protein interactions thus provide potential targets for therapeutic intervention in many disease states. One method known in the art for detecting protein—protein interaction is the yeast two-hybrid system (Fields and Song, 1989, Nature 340: 245-6; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9578-9582). This system is a powerful tool for genetic detection and isolation of yeast cells that harbor cDNA clones encoding interacting proteins. The two-hybrid system permits one to isolate from complex cDNA libraries a single cDNA encoding a protein, referred to as the prey, that interacts with a known protein, termed the bait. Moreover, by use of certain two-hybrid reporter assays one can genetically select for loss-of-interaction mutations in either of the corresponding cDNAs (i.e., either bait or prey). This last feature, incorporated into so called reverse two-hybrid selection protocols (Vidal et al., 1996, Proc. Natl. Acad. Sci. USA 93: 10315-20; Vidal et al., 1996, Proc. Natl. Acad. Sci. USA 93: 10321-26), provides an elegant, powerful and facile means of mapping amino acid determinants of interaction for each of the interacting proteins.
The classical two-hybrid method and derivatives (Durfee et al., 1993, Genes and Dev. 7: 555-569; Gyuris et al., 1993, Cell 75: 791-803; Vojtek et al., 1993 Cell 74: 205-214; Le Douarin et al., 1995, Nucleic Acids Research 23: 876-878; Brent et al., 1997, Annu. Rev. Genet. 31: 663-704; Kolanus, 1999, Curr. Top. Microbiol. Immunol. 243: 37-54; Cagney et al., 2000, Methods in Enzymology 328(C): 3-14; Fashena et al., 2000, Methods in Enzymology 328(C): 14-26) utilize a bait protein fused to a site-specific DNA binding domain and prey proteins fused to a transcriptional activation domain. Bait-prey interaction at the DNA site located within the promoter region of a reporter gene in the yeast nucleus places the transcriptional activation domain in a position to recruit RNA polymerase II (PolII) to the promoter. Thus, the central feature of all of these methods is transcription activator reconstitution (TAR) for Pol II by the two-hybrid protein—protein interaction in the nucleus. Because protein—protein interaction is required to reconstitute the PolII complex on the promoter, expression of a Pol II transcription-dependent reporter gene permits detection of the protein—protein interaction in the host yeast cell.
The two-hybrid system is more sensitive than other techniques known in the art, and is capable of detecting interactions not detectable by other methods such as co-immunoprecipitation (Li et al., 1993, FASEB J. 7: 957-963). The sensitivity of the two-hybrid system is likely due to the fact that transient interactions are sufficient to trigger a small amount of transcription resulting in mRNAs that undergo repeated rounds of translation. This results in a first amplification step (transcription) followed by a second signal output amplification step (translation) when the relatively stable protein is produced. The protein produced catalytically produces the final product that is detected, for example, as yeast colony growth or detectable reporter gene expression resulting from the two-hybrid protein—protein interaction.
Despite this sensitivity, all of the highly similar RNA Polymerase II transcription activator reconstitution (Pol II TAR)-based two-hybrid methods have serious limitations. One serious limitation is that these methods are not at all useful for a very large number of proteins including, but not limited to, virtually all transcriptional activators, transcriptional repressors, RNA polymerase II components, components of the general (basal) transcription machinery (RNA polymerase-associated proteins; >70 proteins), and the very large number of proteins identified as being associated with chromatin or involved in chromatin remodeling. Conventional two-hybrid methods are unavailing in analyzing protein—protein interactions with such proteins because they can activate reporter genes through either direct binding to the RNA polymerase or through binding to other proteins that in turn bind to an RNA polymerase subunit. In addition, practice has shown that multiple proteins not known to be involved in transcription activation or repression mechanisms for Pol II promoters have been found to have intrinsic transcription activation activity in the two-hybrid assay. Consequently numerous proteins cannot be employed as bait for screening cDNA two-hybrid libraries using a Pol II TAR-based method.
Attempts to overcome this limitation have been made in the prior art. For example, a two-hybrid method based on reconstitution of a transcription activator for RNA polymerase III promoters has recently been developed (Marsolier et al., 1999, Methods in Enzymology 303: 411-422). This Pol III TAR-based two-hybrid method capitalizes on the fact that the transcription of yeast class III genes (which include SNR6, tRNA and 5S RNA genes) relies only on two general transcription factors: TFIIIB and TFIIIC. TFIIIC recognizes class III gene promoter elements and assembles TFIIID on the promoter, allowing TFIIIC to recruit RNA polymerase III, which transcribes the gene. The system is cumbersome, however, because it requires two DNA sites: the first one being the A site that specifies transcription initiation mediated by TFIIIB recruitment of RNA Pol III; and the second site being the native binding site for TFIIIC (or B site) located down stream beyond the coding region (for SNR6). This Pol III TAR-based method has not been adopted in the general research community due to its cumbersome features and several additional limitations. Like the Pol II TAR-based method, bait and prey proteins must interact in the nucleus (see below). Only one reporter gene, the UASg-SNR6, is available, making it difficult to eliminate spurious false positives, for example due to mutations in the reporter gene itself. For quantifying the strength of the interaction in this system one must quantify the level of UASg-SNR6 transcripts by northern blots, which is a tedious assay compared, for example, to a colony growth assay.
Another serious limitation of the classical Pol II TAR-based system (and one that is not overcome using the Pol III-TAR based system) is that nuclear localization is required for TAR based two-hybrid methods. Perforce, a protein that normally never enters the nucleus and requires modification in the cytoplasm for proper activity (including binding activity) is not likely to participate in its normal protein interaction(s) if sequestered in the nucleus. For example, post-translational modification (such as phosphorylation) of cytoplasmic proteins is often mediated by interaction with one or more cytoplasmically-confined proteins; this process is illustrated in the many well-established membrane-based receptor signaling cascades. Such modifications would not occur (or would occur only very inefficiently) for proteins targeted to the nucleus in the Pol II and Pol III TAR-based two-hybrid systems known in the art. Thus, the Pol II TAR, Pol III TAR, and all other methods in the art requiring nuclear localization of bait and prey proteins are not useful for detecting protein—protein interactions in a significant fraction of cellular proteins.
Additionally, Pol II-based methods are hampered by a high frequency of false-positives (as shown in Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9578-9582; Brent et al., 1997, Annu. Rev. Genet. 31: 663-704; Serebriiskii et al., 1999, J. Biol. Chem. 274: 17080-17087; and Serebriiskii et al., 2000, Biotechniques 28: 328-336). It is expected that some of these false positives are a consequence of crowded conditions in the nucleus or in proximity to the chromatin, where the interaction must occur.
Two yeast two-hybrid methods not requiring nuclear localization of bait and prey proteins have been disclosed in the art: the split ubiquitin assay and hSos/Ras recruitment assay methods. The split ubiquitin assay utilizes the behavior of free (split) amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) halves of ubiquitin, which can associate to form a native conformation that is specifically cleaved by ubiquitin-specific proteases (UBPs). The unassociated N-terminal or C-terminal half-molecules cannot be recognized or cleaved by the UBPs. In this system, cDNA encoding a bait protein is fused to cDNA encoding the N-terminus or the C-terminal half of a ubiquitin-reporter (R-URA3) fusion construct. Prey cDNA (or a multiplicity of prey library cDNAs) are fused to the N-terminus of the N-terminal half of ubiquitin. If no bait-prey interaction occurs in the cell, the URA3 enzyme (which is provided in fusion with the C-terminal half of ubiquitin) is catalytically active and confers growth-arrest on the cell in the presence of the suicide substrate analogue, 5-fluorotic acid (5-FOA). If bait-prey protein interaction does occur and brings the N-terminal and C-terminal halves of ubiquitin together in the correct steric orientation, cleavage by UBPs occurs, resulting in the URA3 enzyme being cleaved off and rapidly degraded by the N-end rule pathway. In the event of bait-prey interaction, consequently, no URA3 enzyme is present in the cell to convert 5-FOA to a toxic product, and the cells grow to form a detectable colony (see Johnsson et al., 1994, Proc. Natl. Acad. Sci. USA 91: 10340-10344; Stagler et al., 1998, Proc. Natl. Acad. Sci. USA 95: 5187-5192; and Laser et al., 2000, Proc. Natl. Acad. Sci. USA 97: 13732-13737).
The split-ubiquitin assay also has a number of serious limitations, however. One limitation is that the signal output (cell growth in the presence of the toxic precursor, 5-FOA) by which the interaction is detected is dependent on both the interaction of two proteins and a second kinetic step, cleavage of ubiquitin. This second, kinetic, step, is highly sensitive to steric effects arising from sub-optimal positioning or orientation of the N-terminal and C-terminal halves of ubiquitin. Because of the second step and its sensitivity to many factors, the signal output from the split-ubiquitin system cannot be a reliable indication of the strength (as opposed to the existence) of the protein—protein interaction. Weak signal output may be caused by strong interaction between the bait and prey that is unrecognized because of the lack of proportionality between the strength of the interaction and the strength of the signal generated. A second limitation is that any cDNA clones whose overexpression confers resistance to 5-FOA will be falsely identified as positive. It is known in the art that cDNAs encoding small molecule transporters are detected as false-positives because of their ability to confer resistance to 5-FOA (Laser et al., 2000, Proc. Natl. Acad. Sci. USA 97: 13732-13737). A third limitation results from the fact that at high local concentration the N-terminal and C-terminal halves of ubiquitin will associate with one another by themselves irrespective of bait and prey interaction. Thus, although the split-ubiquitin assay measures co-local concentrations of N-terminal and C-terminal halves of ubiquitin (conjugated with prey and bait proteins, respectively), detection is not necessarily a direct result of interaction between bait and prey proteins (Laser et al., 2000, Proc. Natl. Acad. Sci USA 97: 13732-13737). A fourth limitation of the split-ubiquitin method is its lack of signal amplification. A protein—protein interaction that orients the split halves of ubiquitin properly to be cleaved results in the degradation of one URA3 enzyme molecule. Additivity over multiple molecule degradations leads to protection of the cell from 5-FOA toxicity. There is no signal amplification (such as occurs in other two-hybrid systems, typically due to multiple rounds of translation of each mRNA molecule and multiple catalytic turnovers of the HIS3, URA3 or LacZ encoded enzymes in the Pol II- and Pol III-based methods described above). As a result, the split-ubiquitin assay is unlikely to be sensitive enough to detect weak protein—protein interactions that often drive important and dynamic biological processes.
The hSos-recruitment method utilizes hSos, the mammalian Ras guanyl nucleotide exchange factor (Ras GEF), that can complement a temperature sensitive yeast cdc25 mutant having a Ras protein that remains in the GDP-bound inactive form at 36° C. The yeast CDC25 gene encodes the yeast RasGEF (ySos) ortholog, and the cdc25 mutant RasGEF cannot activate Ras at the non-permissive temperature (36° C.). At that temperature the Ras signaling pathway is defective and the cells do not grow well. Introduction of hSos restores Ras pathway signaling and normal growth, providing that it is recruited to the yeast plasma membrane (the site of RasGEF activity). However, recruitment to the yeast membrane occurs only if a bait protein fused to hSos interacts with a membrane-localized prey protein.
In the performance of the hSos-recruitment method, prey proteins are fused to the Src myristoylation signal (Petitjean et al., 1990, Genetic 124: 797-806; Aronheim et al., 1997, Mol. Cell. Biol. 17: 3094-102; and Aronheim et al., 2000, Methods in Enzymology 328: 47-58), resulting in membrane anchorage. In the practice of this method, a library of cDNAs fused to the v-Src myristoylation signal sequence (Myr) is used to transform a yeast host strain that bears the temperature sensitive cdc25-5 allele and is unable to grow well at 36° C.; an example of such a yeast strain is LRA26 (Petitjean et al., 1990, Genetics 124: 797-806). If LRA26 is transformed with a library plasmid encoding a Myr-fusion protein (prey) that interacts with the bait protein (bait-hSos fusion protein), the bait-hSos protein will be recruited to the membrane, resulting in restoration of Ras pathway signaling pathway and improved cell growth at 36° C.
Despite these features, the hSos/Ras recruitment assay itself has a very serious handicap: restoration of normal growth of the temperature-sensitive Ras mutant cells at 36° C. can occur in a great many ways other than via the desired protein—protein interaction. The resulting very high frequency of false positives is due to: 1) the high complexity of the Ras-cAMP signaling pathway downstream of the defective Ras step; and 2) selection of mammalian Ras family members when present on multi-copy plasmids (i.e., overexpressed) in yeast (Aronheim et al., 1997, Mol. Cell. Biol. 17: 3094-102). Multiple copies (i.e., overexpression) of some of the plurality of genes encoding Ras-cAMP pathway proteins, or the occurrence of recessive or dominant yeast chromosomal gene mutations in some of the corresponding genes produce false-positives by restoring normal growth independent of yeast Ras reactivation by hSos. These genes include the adenylate cyclase gene (CYR1), a suppressor of Ras mutation (CAP/SRV2), protein kinase A subunit genes (BCY1, TPK1, TPK2, or TPK3), cAMP-high and low affinity phosphodiesterases (PDE1 or PDE2), G-protein-coupled receptor system for glucose stimulation of cAMP synthesis (GPR1, GPR2) or any genes involved in signaling below Protein Kinase A.
This high background rate severely limits use of the hSos/Ras recruitment assay, if only because additional screening steps are required to identify false positives. In addition to false positives, the hSos/Ras CytoTrap system also produces a low number of independent colonies when used in a single library screening. The additional steps that have been incorporated to minimize these weaknesses add time and effort to the procedure (Huang, W., et al., 2001, Biotechniques 30: 94-100). An additional limitation of the hSos/Ras recruitment assay is the lack of a means of utilizing it in the reverse two-hybrid mode.
Given the above limitations of existing non Pol(II)TAR systems, there is a need in the art for systems that permit identification of cytoplasmic proteins that do not suffer from the intrinsic high background and false positive rates in these prior art methods. Considering the highly regulated nature of most important genes studied to date and the fact that the estimated number of protein-coding sequences in the human genome will be 40,000 to 80,000, it is important not to exclude a large fraction of genes from protein—protein interaction analyses, as occurs using the methods known in the art. In view of the large number of genetic sequences being determined, there is a need in the art for methods of identifying and characterizing the properties of the protein products encoded thereby.